Cytotoxic Effects of Ardisiacrispin A from Labisia pumila on A549 Human Lung Cancer Cells

Background: Lung cancer is the predominant cause of cancer-related fatalities. This prompted our exploration into the anti-lung cancer efficacy of Labisia pumila, a species meticulously selected from the preliminary screening of 600 plants. Methods: Through the strategic implementation of activity-guided fractionation, ardisiacrispin A (1) was isolated utilizing sequential column chromatography. Structural characterization was achieved employing various spectroscopic methods, including nuclear magnetic resonance (NMR), mass spectrometry (MS), and infrared spectroscopy (IR). Results: L. pumila 70% EtOH extract showed significant toxicity in A549 lung cancer cells, with an IC50 value of 57.04 ± 10.28 µg/mL, as well as decreased expression of oncogenes and induced apoptosis. Compound 1, ardisiacrispin A, induced a 50% cell death response in A549 cells at a concentration of 11.94 ± 1.14 µg/mL. Conclusions: The present study successfully investigated ardisiacrispin A extracted from L. pumila leaves, employing a comprehensive spectroscopic approach encompassing NMR, IR, and MS analyses. The anti-lung cancer efficacy of ardisiacrispin A and L. pumila extract was successfully demonstrated for the first time, to the best of our knowledge.


Introduction
Cancer obviously represents one of the biggest challenges to global human health [1].Lung cancer is the predominant cause of cancer-related fatalities on a worldwide scale [2].In 2020, lung cancers constituted 11.4% of newly diagnosed cancer cases, placing the lung as the second most prevalent site of incident cancers, and the majority of cases, approximately 85%, were attributed to a group of histological subtypes collectively known as non-smallcell lung cancer [2,3].Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors such as gefitinib and erlotinib are typically the first chemotherapy treatments for lung cancer.Unfortunately, the prognosis of advanced and recurrent lung cancer remains suboptimal, and standard treatments utilizing cytotoxic anticancer drugs demonstrate limited therapeutic effectiveness [4].In the current landscape of cancer management, diverse treatment options such as chemotherapy, surgery, and radiotherapy exist [5,6].Despite their efficacy, these therapeutic interventions are often associated with severe side effects, posing a substantial risk to patients, or imposing exacting prerequisites for their implementation.
Given the imperative to advance anti-lung cancer drug development, we conducted a screening of potential candidates from a repository of plants within our institute (data not presented).Labisia pumila (Myrsinaceae), grown in southeast Asia, emerged as a promising anti-lung cancer candidate in our preliminary investigation [7].Traditionally employed for the maintenance of female reproductive health and postpartum care, this botanical Life 2024, 14, 276 2 of 10 specimen holds promise in anti-cancer applications.The investigation of this plant has revealed a number of secondary metabolites, exhibiting phytoestrogenic, anti-bacterial, anti-fungal, anti-oxidant, anti-carcinogenic, and anti-aging effects [7][8][9].Historical uses of this botanical entity include the enhancement of stamina and treatment for various conditions, such as dysentery, rheumatism, gonorrhea, excessive flatulence, and cancers, particularly those affecting the breast and uterus [10,11].Despite the well-established anticancer properties of L. pumila, limited information is available concerning its efficacy in the context of lung cancer.Therefore, in the present study, the natural product, L. pumila, and its active compound were rigorously investigated to assess their potential as a candidate for lung cancer treatment.

Reagents and Instrumentation
The equipment and chemicals for isolation and structural elucidation of the anticancer component were referred to in our previous investigations [11,12].Briefly, SiO 2 (Kieselgel 60, Merck, Darmstadt, Germany) and ODS (Lichroprep RP-18, 40-60 µm, Merck) were used as resins for column chromatography (c.c.).The separated compound was detected using a UV lamp (Spectroline Model ENF-240 C/F, Spectronics Corporation, Westbury, NY, USA) following application on Kieselgel 60 F254 (Merck) and Kieselgel RP-18 F254S (Merck) plates, subsequent to spraying with a 10% aqueous H 2 SO 4 solution.Nuclear magnetic resonance (NMR) spectra were recorded employing a Bruker Avance 600 (Billerica, MA, USA), and melting points were precisely determined using a Fisher-John Melting Point Apparatus (Fisher Scientific, Miami, FL, USA).Deuterium solvents for measurement of NMR and standard organic solvents for extraction were purchased from Sigma Aldrich Co., Ltd.(St. Louis, MO, USA) and Daejung Chemical Ltd.(Seoul, Republic of Korea), respectively.

Plants
In this study, a comprehensive set of 600 plant extract samples was sourced from the International Biological Material Research Center (IBMRC, Cheongju, Republic of Korea).Voucher specimens, uniquely identified by the codes KHU-BMRI-2017-001 through KHU-BMRI-2017-600, were deposited at the Bio-Medical Research Institute, Yongin, Kyung Hee University.The process for isolating potential anti-cancer candidates involved the extraction of plant materials using 100% methanol (MeOH), and the extracts were subsequently solubilized to a concentration of 10 mg/mL in dimethylsulfoxide (DMSO).

Cell Viability and Cytotoxicity Assay
A549 human cell lines, obtained from the Korean Cell Line Bank (KCLB), were cultured in RPMI1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin [13].The cells were cultivated in a humidified incubator at 37 • C with a CO 2 concentration of 5%.Cell viability was evaluated utilizing the MTT assay, with seeding densities of 5 × 10 3 cells/well in a 96-well plate.Following a 24 h incubation period, the culture medium was replaced, and the samples were subjected to experimental treatments.After 24 h, the cells were stained using MTT solution in PBS, resulting in a final concentration of 0.5 mg/mL.The cells were subjected to a 4 h incubation at 37 • C. Upon completion of this incubation period, the supernatant was removed and 100 µL of DMSO was added.Utilizing a microplate reader (Tecan, Switzerland), absorbance was measured at 540 nm.The cell cytotoxicity rates were calculated based on the optical density readings, expressed as percentages relative to the vehicle control, and this procedure was repeated for accuracy.

Tali TM Cell Cycle Assay
Human A549 cells were seeded in 6-well plates at a density of 1 × 10 5 cells/well.After 24 h incubation, the culture medium was replaced, and the cells were subjected to specific treatments.Following fixation, the cells were treated with the optimized Tali TM cell cycle reagent (Thermo, Middlesex, MA, USA) and incubated in darkness for 30 min.The Tali ® image cytometer (Thermo, Middlesex, MA, USA) was employed for cell cycle analysis.The acquired cell cycle data from the Tali TM image-based cytometer were analyzed both on the instrument and through dedicated cell cycle modeling software.

Statistical Analysis
All data are presented as mean ± standard error of the mean (SEM).The significance of differences between groups was assessed using one-way analysis of variance (ANOVA).Statistical significance was defined as p < 0.05.

Determination of the Anti-Cancer Agent and Its Optimal Extraction Condition
In pursuit of identifying a natural anti-cancer candidate agent, a pilot study was conducted through cell viability assays to determine IC 50 values in A549 cells.From an extensive pool of MeOH extracts obtained from a diverse collection of over 600 plants, L. pumila emerged as a potent anti-lung cancer candidate.Following this selection, a systematic evaluation was carried out to establish the optimal concentration of the L. pumila extract.The evaluation revealed the significant toxicity demonstrated by the 70% EtOH extract in A549 lung cancer cells, with an IC 50 value of 57.04 ± 10.28 µg/mL (Table 1).The dried leaves of L. pumila were subjected to extraction using 70% aqueous EtOH, and the resulting concentrate was fractioned into n-hexane (LPH), dichloromethane (LPD), ethyl acetate (LPE), n-BuOH (LPB), and H 2 O (LPW) fractions.A series of activity-guided fractionation steps, employing SiO 2 , ODS, and Sephadex LH-20 column chromatography (c.c.) for the LPD fraction, led to the isolation of a singular triterpenoid saponin (1).Elucidation of its chemical structure was achieved through a comprehensive analysis of spectroscopic data, including mass spectrometry (MS), infrared spectroscopy (IR), and NMR (both 1D and 2D).

Regulation of Cell Cycle by L. pumila
Apoptosis, an intricate mechanism of programmed cell death inherent to multicellular organisms, serves as a pivotal process for the elimination of undesirable and defective cells [19].This orchestrated cellular death not only facilitates the removal of superfluous entities but also mitigates the risk of inciting undesirable inflammatory responses.Apoptosis is a ubiquitous phenomenon, actively participating during normal development and cellular turnover, as well as extending to various pathological conditions.
The cell cycle, a highly conserved mechanism, orchestrates the replication of eukaryotic cells.The regulation of cell death is intricately linked to genes governing cell cycle

Regulation of Cell Cycle by L. pumila
Apoptosis, an intricate mechanism of programmed cell death inherent to multicellular organisms, serves as a pivotal process for the elimination of undesirable and defective cells [19].This orchestrated cellular death not only facilitates the removal of superfluous entities but also mitigates the risk of inciting undesirable inflammatory responses.Apoptosis is a ubiquitous phenomenon, actively participating during normal development and cellular turnover, as well as extending to various pathological conditions.
The cell cycle, a highly conserved mechanism, orchestrates the replication of eukaryotic cells.The regulation of cell death is intricately linked to genes governing cell cycle progression.Cumulative evidence has underscored the impact of cell cycle manipulation on modulating apoptosis reactions, contingent upon the specific cellular context [20,21].
Concentration-dependent reductions in the G0/G1, G2/M, and S phases were discerned in response to the L. pumila extract.Conversely, an elevation in the Sub G1 phase was observed upon treatment with the L. pumila extract.It is well documented that an augmentation in the Sub G1 phase corresponds to the onset of apoptosis.Thus, the observed elevation in the Sub G1 phase following treatment with the L. pumila extract can be attributed to the induction of apoptosis (Figure 2).
progression.Cumulative evidence has underscored the impact of cell cycle manipulation on modulating apoptosis reactions, contingent upon the specific cellular context [20,21].
Concentration-dependent reductions in the G0/G1, G2/M, and S phases were discerned in response to the L. pumila extract.Conversely, an elevation in the Sub G1 phase was observed upon treatment with the L. pumila extract.It is well documented that an augmentation in the Sub G1 phase corresponds to the onset of apoptosis.Thus, the observed elevation in the Sub G1 phase following treatment with the L. pumila extract can be attributed to the induction of apoptosis (Figure 2).
Extracellular signal-regulated kinase (ERK) plays a crucial role in tumorigenesis [27].ERK activity is associated with the promotion of apoptotic pathways, including the induction of mitochondrial cytochrome C release, caspase-8 activation, permanent cell cycle arrest, and autophagic vacuolization.The active state of ERK is characterized by its phosphorylated form [28][29][30]. Figure 4 demonstrates that the p-ERK/ERK ratio in ardisiacrispin A-treated cells was lower than the normal control.In addition, L. pumila revealed a dosedependent decrease in the p-ERK/ERK ratio.Extracellular signal-regulated kinase (ERK) plays a crucial role in tumorigenesis [27].ERK activity is associated with the promotion of apoptotic pathways, including the induction of mitochondrial cytochrome C release, caspase-8 activation, permanent cell cycle arrest, and autophagic vacuolization.The active state of ERK is characterized by its phosphorylated form [28][29][30]. Figure 4 demonstrates that the p-ERK/ERK ratio in ardisiacrispin A-treated cells was lower than the normal control.In addition, L. pumila revealed a dosedependent decrease in the p-ERK/ERK ratio.

Conclusions
The present study successfully extracted ardisiacrispin A (1) from L. pumila leaves, employing a comprehensive spectroscopic approach encompassing NMR, IR, and MS analyses.Compound 1 was isolated from L. pumila leaves for the first time in this study.L. pumila and its active compound, ardisiacrispin A (1), demonstrated potential in sup-  Extracellular signal-regulated kinase (ERK) plays a crucial role in tumorigenesis [27].ERK activity is associated with the promotion of apoptotic pathways, including the induction of mitochondrial cytochrome C release, caspase-8 activation, permanent cell cycle arrest, and autophagic vacuolization.The active state of ERK is characterized by its phosphorylated form [28][29][30]. Figure 4 demonstrates that the p-ERK/ERK ratio in ardisiacrispin A-treated cells was lower than the normal control.In addition, L. pumila revealed a dosedependent decrease in the p-ERK/ERK ratio.

Conclusions
The present study successfully extracted ardisiacrispin A (1) from L. pumila leaves, employing a comprehensive spectroscopic approach encompassing NMR, IR, and MS analyses.Compound 1 was isolated from L. pumila leaves for the first time in this study.L. pumila and its active compound, ardisiacrispin A (1), demonstrated potential in sup-

Conclusions
The present study successfully extracted ardisiacrispin A (1) from L. pumila leaves, employing a comprehensive spectroscopic approach encompassing NMR, IR, and MS analyses.Compound 1 was isolated from L. pumila leaves for the first time in this study.L. pumila and its active compound, ardisiacrispin A (1), demonstrated potential in suppressing the proliferation and metastasis of lung cancer cells.This inhibitory effect is attributed to the modulation of oncogenic signaling pathways related to EGFR and FGER in lung cancer.Although further apoptosis studies are needed to investigate the involved apoptotic pathways, this is the first report to demonstrate the anti-lung cancer efficacy of ardisiacrispin A (1) and L. pumila extract, to the best of our knowledge.Consequently, these findings underscore the feasibility of utilizing ardisiacrispin A (1) and L. pumila extract as antilung cancer agents.To validate their efficacy as anti-lung cancer candidates, further investigations encompassing the elucidation of the mode of action and preclinical trials are imperative.

Figure 4 .
Figure 4. Changes in protein expression levels (p-ERK and ERK) associated with cell cycle regulation.Data are presented as the mean ± standard deviation.n = 3, ** p < 0.01.

Figure 4 .
Figure 4. Changes in protein expression levels (p-ERK and ERK) associated with cell cycle regulation.Data are presented as the mean ± standard deviation.n = 3, ** p < 0.01.

Figure 4 .
Figure 4. Changes in protein expression levels (p-ERK and ERK) associated with cell cycle regulation.Data are presented as the mean ± standard deviation.n = 3, ** p < 0.01.